1 Introduction

Macrophages are mononuclear cells with high phagocytic activity, (Schnyder and Baggiolini 1978). They have key roles in the immune responses, particularly the production of proinflammatory cytokines and the phagocytosis of microorganisms, (Murray et al. 2005).

Phagocytosis is a major function of the macrophage, not only for pathogens, but also for the resolution of inflammation through the phagocytosis of other immune cells, such as neutrophils, (Newman, Henson, and Henson 1982).

The necrosis of tumours in response to bacterial-endotoxins was first determined to be an indirect process by, (O’Malley, Achinstein, and Shear 1962), in which the necrosis could be triggered by a serum, derived from animals treated with lipopolysaccharides (LPS), in animals not exposed to LPS. This was deemed to be mediated by a “tumour necrotizing factor” later determined to be a superfamily that includes the protein Tumour Necrosis Factor-\(\alpha\) (TNF-\(\alpha\)).

Understanding the mechanisms by which macrophages are activated, and their subsequent production of TNF-\(\alpha\), is important for aiding research into chronic inflammatory diseases such as rheumatoid arthritis and multiple sclerosis, (Plevy et al. 1997). Uncontrolled macrophage activation is noted to be key in such chronic inflammatory diseases, (Sindrilaru et al. 2011), with TNF-\(\alpha\) being classed as a proinflammatory macrophage marker.

The aim of this study is to gain understanding of the mechanisms by which macrophages are activated/respond and produce TNF-\(\alpha\), specifically with regards to the necessity of phagocytosis in the production of TNF-\(\alpha\).

2 Is phagocytosis required to trigger the production of TNF-\(\alpha\) by mcrophages?

The TNF-\(\alpha\) production from RAW 264.7 cells is shown in Figures 2.1 and 2.2. Membranes from the SDS-PAGE were incubated with the primary antibody solution, rabbit anti-TNF-\(\alpha\) in TBS-T with 5% milk, and consequently incubated with the secondary antibody solution, goat anti-rabbit IgG-linked to horseradish peroxidase in PBS-T in milk.

All cells were treated with Brefeldin-A, an inhibitor of protein transport and secretion, in order to enhance the intracellular cytokine (TNF-\(\alpha\)) staining signal for the Western Blot Analysis.

Figure 2.1 indicates that only RAW 264.7 cells treated with 100 ng/ml LPS from E.coli and RAW 264.7 cells treated with E.coli bacteria produced TNF-\(\alpha\), as shown by the bands labelled B1, B2, C1 and C2 respectively.

Figure 2.2, illustrates the calculation of the molecular weights of the labelled bands from Figure 2.1, using the calculated molecular weights of the molecular marker bands. From the calculated molecular weights of Figure 2.2, the identity of the bands on the Western Blot can be confirmed to be that of TNF-\(\alpha\).

Bands B1 and C1 calculated to be 28.3 kDa and 27.11 kDa respectively. Bands B2 and C2 calculated to be 22.9 kDa and 23.89 kDa respectively. These were compared to the datasheet, (CellSignal 2017) and (UniProtConsortium 2019), to confirm the identification of the TNF-\(\alpha\) bands.

Western blot from SDS-PAGE, analysis of TNF-\(\alpha\) in RAW 264.7 cells treated with Brefeldin-A. (A) Untreated RAW 264.7 cells (B) RAW 264.7 cells treated with 100 ng/ml LPS from E.coli (C) RAW 264.7 cells treated with E.coli bacteria

Figure 2.1: Western blot from SDS-PAGE, analysis of TNF-\(\alpha\) in RAW 264.7 cells treated with Brefeldin-A.
(A) Untreated RAW 264.7 cells
(B) RAW 264.7 cells treated with 100 ng/ml LPS from E.coli
(C) RAW 264.7 cells treated with E.coli bacteria

Identification of the Molecular Weight of TNF-\(\alpha\) using Rf values Dashed Red - B1 band shown in Figure 2.1, Red - B2 band shown in Figure 2.1, Dashed Blue - C1 band shown in Figure 2.1, Blue - C2 band shown in Figure 2.1.

Figure 2.2: Identification of the Molecular Weight of TNF-\(\alpha\) using Rf values
Dashed Red - B1 band shown in Figure 2.1, Red - B2 band shown in Figure 2.1, Dashed Blue - C1 band shown in Figure 2.1, Blue - C2 band shown in Figure 2.1.

3 Determining the level of TNF-\(\alpha\) expression using Flow Cytometry

The western blotting results looked at the overall effect of LPS and bacteria on a population of cells, without distinction between individual cells. Flow cytometry can be used to analyse the production of TNF-\(\alpha\) by individual RAW 264.7 macrophage cells.

3.1 Principles of Flow Cytometry

Flow cytometry involves a cell susupension being passed through a flow chamber, so that cells pass in front of lasers one-by-one. This results in light being scattered and detected by forward and side scatter detectors, allowing the determination of size and granularity. Fluorescence detectors also detect any fluorescently tagged cells for identification of specific markers.

For an overview of the technique please see the video, (mitedustar 2015).

4 Methods

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##      X12    X18 X21
## [1,]   1 0.0727   0
## [2,]   0 1.0000   0
## [3,]   0 0.0000   1
## $SPILL
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## $`$SPILLOVER`
##      X12    X18 X21
## [1,]   1 0.0727   0
## [2,]   0 1.0000   0
## [3,]   0 0.0000   1
##      X12    X18 X21
## [1,]   1 0.0727   0
## [2,]   0 1.0000   0
## [3,]   0 0.0000   1
## $SPILL
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## $spillover
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##      X12    X18 X21
## [1,]   1 0.0727   0
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##      X12    X18 X21
## [1,]   1 0.0727   0
## [2,]   0 1.0000   0
## [3,]   0 0.0000   1
## $SPILL
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## [1,]   1 0.0727   0
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## [3,]   0 0.0000   1

5 Results

References

mitedustar. 2015. “Flow Cytometry Animation.” Youtube. https://www.youtube.com/watch?v=EQXPJ7eeesQ.

Murray, Rachael Z, Jason G Kay, Daniele G Sangermani, and Jennifer L Stow. 2005. “A Role for the Phagosome in Cytokine Secretion.” Science 310 (5753): 1492–5. https://doi.org/10.1126/science.1120225.

Newman, S L, J E Henson, and P M Henson. 1982. “Phagocytosis of Senescent Neutrophils by Human Monocyte-Derived Macrophages and Rabbit Inflammatory Macrophages.” J. Exp. Med. 156 (2): 430–42. https://doi.org/10.1084/jem.156.2.430.

O’Malley, W Edward, Betty Achinstein, and Murray J Shear. 1962. “Action of Bacterial Polysaccharide on Tumors. II. Damage of Sarcoma 37 by Serum of Mice Treated with Serratia Marcescens Polysaccharide, and Induced Tolerance.” J. Natl. Cancer Inst. 29 (6): 1169–75. https://doi.org/10.1093/jnci/29.6.1169.

Plevy, S E, C J Landers, J Prehn, N M Carramanzana, R L Deem, D Shealy, and S R Targan. 1997. “A Role for TNF-alpha and Mucosal T Helper-1 Cytokines in the Pathogenesis of Crohn’s Disease.” J. Immunol. 159 (12): 6276–82. https://www.ncbi.nlm.nih.gov/pubmed/9550432.

Schnyder, J, and M Baggiolini. 1978. “Role of Phagocytosis in the Activation of Macrophages.” J. Exp. Med. 148 (6): 1449–57. https://doi.org/10.1084/jem.148.6.1449.

Sindrilaru, Anca, Thorsten Peters, Stefan Wieschalka, Corina Baican, Adrian Baican, Henriette Peter, Adelheid Hainzl, et al. 2011. “An Unrestrained Proinflammatory M1 Macrophage Population Induced by Iron Impairs Wound Healing in Humans and Mice.” J. Clin. Invest. 121 (3): 985–97. https://doi.org/10.1172/JCI44490.

UniProtConsortium. 2019. “Tnf - Tumor Necrosis Factor Precursor - Mus Musculus (Mouse) - Tnf Gene & Protein.” https://www.uniprot.org/uniprot/P06804. https://www.uniprot.org/uniprot/P06804.